Polymorphic chromosomal inversions in Anopheles moucheti,a major malaria vector in Central Africa

Anopheles moucheti Evans (Diptera: Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on the 2R chromosome arm. The homology between the 2R chromosome arms of An. moucheti and Anopheles gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new basis for further population genetic, taxonomic and ecological studies of this neglected malaria vector.     

Chromosomal evidence of incipient speciation in the Afrotropical malaria mosquito Anopheles funestus

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.     

Association mapping desiccation resistance within chromosomal inversions in the African malaria vector Anopheles gambiae

Inversion polymorphisms are responsible for many ecologically important phenotypes and are often found under balancing selection. However, the same features that ensure their large role in local adaptation—especially reduced recombination between alternate arrangements—mean that uncovering the precise loci within inversions that control these phenotypes is unachievable using standard mapping approaches. Here, we take advantage of long‐term balancing selection on a pair of inversions in the mosquito Anopheles gambiae to map desiccation tolerance via pool‐GWAS. Two polymorphic inversions on chromosome 2 of this species (denoted 2La and 2Rb) are associated with arid and hot conditions in Africa and are maintained in spatially and temporally heterogeneous environments. After measuring thousands of wild‐caught individuals for survival under desiccation stress, we used phenotypically extreme individuals homozygous for alternative arrangements at the 2La inversion to construct pools for whole‐genome sequencing. Genomewide association mapping using these pools revealed dozens of significant SNPs within both 2La and 2Rb, many of which neighboured genes controlling ion channels or related functions. Our results point to the promise of similar approaches in systems with inversions maintained by balancing selection and provide a list of candidate genes underlying the specific phenotypes controlled by the two inversions studied here.     

Chromosomal inversions and ecotypic differentiation in Anopheles gambiae: the perspective from whole‐genome sequencing

The molecular mechanisms and genetic architecture that facilitate adaptive radiation of lineages remain elusive. Polymorphic chromosomal inversions, due to their recombination‐reducing effect, are proposed instruments of ecotypic differentiation. Here, we study an ecologically diversifying lineage of Anopheles gambiae, known as the Bamako chromosomal form based on its unique complement of three chromosomal inversions, to explore the impact of these inversions on ecotypic differentiation. We used pooled and individual genome sequencing of Bamako, typical (non‐Bamako) An. gambiae and the sister species Anopheles coluzzii to investigate evolutionary relationships and genomewide patterns of nucleotide diversity and differentiation among lineages. Despite extensive shared polymorphism and limited differentiation from the other taxa, Bamako clusters apart from the other taxa, and forms a maximally supported clade in neighbour‐joining trees based on whole‐genome data (including inversions) or solely on collinear regions. Nevertheless, F_ST outlier analysis reveals that the majority of differentiated regions between Bamako and typical An. gambiae are located inside chromosomal inversions, consistent with their role in the ecological isolation of Bamako. Exceptionally differentiated genomic regions were enriched for genes implicated in nervous system development and signalling. Candidate genes associated with a selective sweep unique to Bamako contain substitutions not observed in sympatric samples of the other taxa, and several insecticide resistance gene alleles shared between Bamako and other taxa segregate at sharply different frequencies in these samples. Bamako represents a useful window into the initial stages of ecological and genomic differentiation from sympatric populations in this important group of malaria vectors.     

Rangewide population genetic structure of the African malaria vector Anopheles funestus

Anopheles funestus is a primary vector of malaria in Africa south of the Sahara. We assessed its rangewide population genetic structure based on samples from 11 countries, using 10 physically mapped microsatellite loci, two per autosome arm and the X (N = 548), and 834 bp of the mitochondrial ND5 gene (N = 470). On the basis of microsatellite allele frequencies, we found three subdivisions: eastern (coastal Tanzania, Malawi, Mozambique and Madagascar), western (Burkina Faso, Mali, Nigeria and western Kenya), and central (Gabon, coastal Angola). A. funestus from the southwest of Uganda had affinities to all three subdivisions. Mitochondrial DNA (mtDNA) corroborated this structure, although mtDNA gene trees showed less resolution. The eastern subdivision had significantly lower diversity, similar to the pattern found in the codistributed malaria vector Anopheles gambiae. This suggests that both species have responded to common geographic and/or climatic constraints. The western division showed signatures of population expansion encompassing Kenya west of the Rift Valley through Burkina Faso and Mali. This pattern also bears similarity to A. gambiae, and may reflect a common response to expanding human populations following the development of agriculture. Due to the presumed recent population expansion, the correlation between genetic and geographic distance was weak. Mitochondrial DNA revealed further cryptic subdivision in A. funestus, not detected in the nuclear genome. Mozambique and Madagascar samples contained two mtDNA lineages, designated clade I and clade II, that were separated by two fixed differences and an average of 2% divergence, which implies that they have evolved independently for approximately 1 million years. Clade I was found in all 11 locations, whereas clade II was sampled only on Madagascar and Mozambique. We suggest that the latter clade may represent mtDNA capture by A. funestus, resulting from historical gene flow either among previously isolated and divergent populations or with a related species.     

Chromosome inversions and ecological plasticity in the main African malaria mosquitoes

Chromosome inversions have fascinated the scientific community, mainly because of their role in the rapid adaption of different taxa to changing environments. However, the ecological traits linked to chromosome inversions have been poorly studied. Here, we investigated the roles played by 23 chromosome inversions in the adaptation of the four major African malaria mosquitoes to local environments in Africa. We studied their distribution patterns by using spatially explicit modeling and characterized the ecogeographical determinants of each inversion range. We then performed hierarchical clustering and constrained ordination analyses to assess the spatial and ecological similarities among inversions. Our results show that most inversions are environmentally structured, suggesting that they are actively involved in processes of local adaptation. Some inversions exhibited similar geographical patterns and ecological requirements among the four mosquito species, providing evidence for parallel evolution. Conversely, common inversion polymorphisms between sibling species displayed divergent ecological patterns, suggesting that they might have a different adaptive role in each species. These results are in agreement with the finding that chromosomal inversions play a role in Anopheles ecotypic adaptation. This study establishes a strong ecological basis for future genome‐based analyses to elucidate the genetic mechanisms of local adaptation in these four mosquitoes.     

Mitochondrial and ribosomal internal transcribed spacer (ITS2) diversity of the African malaria vector Anopheles funestus

The pattern of sequence variation in the mitochondrial DNA cytochrome b gene (cyt-b) and ribosomal DNA internal transcribed spacer 2 (ITS2) was examined in Anopheles funestus from Senegal and Burkina Faso in West Africa and Kenya in East Africa. From both West African countries, samples included individuals hypothesized to represent reproductively isolated taxa based upon different karyotypes and behaviours. Analysis of the cyt-b data revealed high haplotypic diversity (86%) and an average pairwise difference per site of 0.42%. Sequence variation was not partitioned by geographical origin or karyotype class. The most common haplotype was sampled across Africa (approximately 6000 km). Analysis of the ITS2 data revealed one of the longest spacers yet found in anophelines (approximately 704 bp). In common with other anopheline ITS2 sequences, this one had microsatellites and frequent runs of individual nucleotides. Also in common with data from other anopheline ITS2 studies, the An. funestus sequences were almost monomorphic, with only two rare polymorphisms detected. The results from both markers are congruent and do not support the hypothesis of reproductively isolated chromosomal taxa within An. funestus. Whether the lack of support by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) sequences is a result of the recent origin of the presumptive taxa, or of the absence of barriers to gene flow, remains to be elucidated, using more rapidly evolving markers such as microsatellites.     

Relative developmental and reproductive fitness associated with pyrethroid resistance in the major southern African malaria vector, Anopheles funestus

The effect of pyrethroid resistance on the fitness of a laboratory strain of Anopheles funestus originating from southern Mozambique was evaluated by comparing the developmental and reproductive characteristics of a pyrethroid resistant strain with an insecticide susceptible strain. Fitness was evaluated in terms of fecundity, fertility, egg production, developmental time and life stage progression and survival. Of the eggs laid by females of the resistant strain, 81.5% hatched while only 66.9% were recorded in the susceptible strain. The time from egg hatch to adult emergence was longer for the resistant strain (15.9 days) than the susceptible strain (15.2 days). A significantly higher proportion of eggs from the resistant strain (61.6%) survived to adulthood compared with those of the susceptible strain (49%). Fecundity and larval and pupal survival did not differ significantly between strains. Of spermathecae dissected from females of the resistant strain, 56.8% were fertilized compared to 52.6% from the susceptible strain. The proportion of females that successfully produced eggs was 43.3% and 23.3% for the resistant and susceptible strains respectively. Complete failure of larval hatch was recorded in 28.6% of susceptible strain families compared to 7.7% of resistant families. Our results show that pyrethroid resistance in southern African An. funestus does not incur any loss of fitness under laboratory conditions. These results suggest that the removal of pyrethroid insecticide selection pressure may not lead to a regression of resistance alleles in pyrethroid resistant An. funestus populations in southern Africa.     

Linkage disequilibrium network analysis (LDna) gives a global view of chromosomal inversions,local adaptation and geographic structure

Recent advances in sequencing allow population‐genomic data to be generated for virtually any species. However, approaches to analyse such data lag behind the ability to generate it, particularly in nonmodel species. Linkage disequilibrium (LD, the nonrandom association of alleles from different loci) is a highly sensitive indicator of many evolutionary phenomena including chromosomal inversions, local adaptation and geographical structure. Here, we present linkage disequilibrium network analysis (LDna), which accesses information on LD shared between multiple loci genomewide. In LD networks, vertices represent loci, and connections between vertices represent the LD between them. We analysed such networks in two test cases: a new restriction‐site‐associated DNA sequence (RAD‐seq) data set for Anopheles baimaii, a Southeast Asian malaria vector; and a well‐characterized single nucleotide polymorphism (SNP) data set from 21 three‐spined stickleback individuals. In each case, we readily identified five distinct LD network clusters (single‐outlier clusters, SOCs), each comprising many loci connected by high LD. In A. baimaii, further population‐genetic analyses supported the inference that each SOC corresponds to a large inversion, consistent with previous cytological studies. For sticklebacks, we inferred that each SOC was associated with a distinct evolutionary phenomenon: two chromosomal inversions, local adaptation, population‐demographic history and geographic structure. LDna is thus a useful exploratory tool, able to give a global overview of LD associated with diverse evolutionary phenomena and identify loci potentially involved. LDna does not require a linkage map or reference genome, so it is applicable to any population‐genomic data set, making it especially valuable for nonmodel species.     

Anopheles arabiensis and An. gambiae chromosomal inversion polymorphism, feeding and resting behaviour in relation to insecticide house-spraying in Tanzania

Abstract. Differential responses of the mosquitoes Anopheles arabiensis and An. gambiae sensu stricto to house-spraying with DDT or lambda-cyhalothrin were evaluated in relation to chromosomal inversion polymorphism, feeding and resting behaviour of these malaria vectors in Tanzania. Blood-fed mosquitoes from pit traps outdoors, exit traps on windows and indoor-resting catches were identified cytogenetically and the chromosomal inversion frequencies compared between samples and species. Their outdoor-resting behaviour was assessed by a mark–release–recapture experiment and by determining the proportion of freshly blood-fed individuals in exit traps. The source of bloodmeals was analysed by an ELISA method. Endophagic females of An. arabiensis were more likely than those of An. gambiae to exit from a house on the night of blood-feeding. Only in one out of three villages was there evidence that chromosomally distinct individuals within a species had different preferences for resting sites. There were indications, but not conclusive evidence, that mosquitoes caught indoors or outdoors had a tendency to return to the same type of resting site. In villages sprayed with either insecticide, the mean age of the vector populations was greatly reduced, compared with those in the unsprayed villages. An. arabiensis females exited from DDT sprayed houses after blood-feeding, whereas with lambda-cyhalothrin those exiting were mostly unfed and there was a decline in the human blood index. The excitorepellency of DDT was perceived as a disadvantage, whereas lambda-cyhalothrin apparently had more impact on malaria transmission by An. arabiensis.     

Advances in the study of Anopheles funestus, a major vector of malaria in Africa

The recent literature on cytogenetic and molecular studies of Anopheles funestus, a major vector of malaria in Africa, is reviewed. Molecular data from West and Central Africa suggest a new species in the group closely allied to Anopheles rivulorum. Cytogenetic and molecular studies of populations from West, Central, East and southern Africa indicate considerable genetic structuring within An. funestus itself, which may well restrict the spread of pyrethroid resistance that has been demonstrated in southern Africa.     

Genetic loads and coadaptivity of chromosomal inversions I. Inversion polymorphism and genetic load on chromosome O in a Drosophila subobscura population from Petnica

Inversion polymorphism on chromosome O and polymorphism for the viability of determining genes have been studied in a natural population of Drosophila subobscura from Petnica (Serbia). The range of inversion polymorphism and the abundance of particular gene arrangements in the study population agree with a general pattern of inversion polymorphism of D. subobscura in Europe. The data obtained on the amount of genetic loads show that the D. subobscura population from Petnica displays a moderate degree of that polymorphism, compared to the other studied populations of these species. Therefore, the D. subobscura population from Petnica could be tentatively classified as an ecologically central population. Examination association of chromosomal, thus, inversion polymorphism with gene polymorphism, in the form of genetic loads show that differences exist in the mean viability among certain gene arrangements. The distribution of deleterious genes among chromosome O gene arrangements were non-random.     

Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa

Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction.Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the ??-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique.The distribution of the Rdl^R mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions.     

Long‐term balancing selection on chromosomal variants associated with crypsis in a stick insect

How polymorphisms are maintained within populations over long periods of time remains debated, because genetic drift and various forms of selection are expected to reduce variation. Here, we study the genetic architecture and maintenance of phenotypic morphs that confer crypsis in Timema cristinae stick insects, combining phenotypic information and genotyping‐by‐sequencing data from 1,360 samples across 21 populations. We find two highly divergent chromosomal variants that span megabases of sequence and are associated with colour polymorphism. We show that these variants exhibit strongly reduced effective recombination, are geographically widespread and probably diverged millions of generations ago. We detect heterokaryotype excess and signs of balancing selection acting on these variants through the species’ history. A third chromosomal variant in the same genomic region likely evolved more recently from one of the two colour variants and is associated with dorsal pattern polymorphism. Our results suggest that large‐scale genetic variation associated with crypsis has been maintained for long periods of time by potentially complex processes of balancing selection.     

Modification of spontaneous activity patterns in the malaria vector Anopheles gambiae sensu stricto when presented with host‐associated stimuli

Anopheles gambiae sensu stricto Giles (Diptera: Culicidae) is the main malaria vector in sub‐Saharan Africa. Mated females show a circadian rhythm of spontaneous activity under constant environmental conditions that extends across the scotophase (subjective night). The effect of host‐associated cues [i.e. human foot odour supplemented with carbon dioxide (CO₂)] on this nocturnal activity pattern is studied in laboratory‐reared A. gambiae s.s. M molecular form. Sixteen mated females (5–10 days old) are held in individual chambers (diameter 3.5 cm, length 4.5 cm) in a wind‐tunnel with a continuous flow (8.0 cm s^?1) of clean air. At the onset of hours 3, 6 and 10 of the scotophase, their behaviour is recorded on video for 15 min in clean moving air and then for the next 15 min, with a specific treatment present in the clean airstream: (i) constant CO₂ (4.8%) plus human odour; (ii) pulsed CO₂ (5 s of every 30 s) plus human odour or (iii) control (clean air). Activities of individual mosquitoes are scored by direct observation of the video records for the incidents of ‘resting’, ‘walking’, ‘jumping’ and ‘flying’ in each of the 15‐min observation periods. There is a significant interaction between hour and treatment on mean changes in female activity level (P = 0.00004). Constant treatment increases the level of activity of A. gambiae females significantly, although only in hour 3 of the scotophase (P < 0.01), whereas pulsed and control treatments show no significant effect throughout the scotophase. Patterns of spontaneous activity in individual A. gambiae females thus appear to be modulated by host‐associated cues, resulting in a more active phase early in the night than might be expected from records of spontaneous activity under constant environmental conditions. Possible ecological and experimental implications of such an increase in activity are discussed in relation to host‐seeking behaviour.     

Induction of telomere‐mediated chromosomal truncation and behavior of truncated chromosomes in Brassica napus

Engineered minichromosomes could be stably inherited and serve as a platform for simultaneously transferring and stably expressing multiple genes. Chromosomal truncation mediated by repeats of telomeric sequences is a promising approach for the generation of minichromosomes. In the present work, direct repetitive sequences of Arabidopsis telomere were used to study telomere‐mediated truncation of chromosomes in Brassica napus. Transgenes containing alien Arabidopsis telomere were successfully obtained, and Southern blotting and fluorescence in situ hybridization (FISH) results show that the transgenes resulted in successful chromosomal truncation in B. napus. In addition, truncated chromosomes were inherited at rates lower than that predicted by Mendelian rules. To determine the potential manipulations and applications of the engineered chromosomes, such as the stacking of multiple transgenes and the Cre/lox and FRT/FLP recombination systems, both amenable to genetic manipulations through site‐specific recombination in somatic cells, were tested for their ability to undergo recombination in B. napus. These results demonstrate that alien Arabidopsis telomere is able to mediate chromosomal truncation in B. napus. This technology would be feasible for chromosomal engineering and for studies on chromosome structure and function in B. napus.     

Genetic structuring in a Neotropical palm analyzed through an Andean orogenesis‐scenario

Andean orogenesis has driven the development of very high plant diversity in the Neotropics through its impact on landscape evolution and climate. The analysis of the intraspecific patterns of genetic structure in plants would permit inferring the effects of Andean uplift on the evolution and diversification of Neotropical flora. In this study, using microsatellite markers and Bayesian clustering analyses, we report the presence of four genetic clusters for the palm Oenocarpus bataua var. bataua which are located within four biogeographic regions in northwestern South America: (a) Chocó rain forest, (b) Amotape‐Huancabamba Zone, (c) northwestern Amazonian rain forest, and (d) southwestern Amazonian rain forest. We hypothesize that these clusters developed following three genetic diversification events mainly promoted by Andean orogenic events. Additionally, the distinct current climate dynamics among northwestern and southwestern Amazonia may maintain the genetic diversification detected in the western Amazon basin. Genetic exchange was identified between the clusters, including across the Andes region, discarding the possibility of any cluster to diversify as a distinct intraspecific variety. We identified a hot spot of genetic diversity in the northern Peruvian Amazon around the locality of Iquitos. We also detected a decrease in diversity with distance from this area in westward and southward direction within the Amazon basin and the eastern Andean foothills. Additionally, we confirmed the existence and divergence of O. bataua var. bataua from var. oligocarpus in northern South America, possibly expanding the distributional range of the latter variety beyond eastern Venezuela, to the central and eastern Andean cordilleras of Colombia. Based on our results, we suggest that Andean orogenesis is the main driver of genetic structuring and diversification in O. bataua within northwestern South America.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.